1. Field of the Invention
The present invention relates generally to the prevention of bovine respiratory disease (BRD) and, in particular, its most severe form, termed “shipping fever”. More specifically, the present invention relates to the use of chimeric proteins comprising immunodominant epitopes of recombinant Mannheimia haemolytica outer membrane protein PlpE, and immunodominant epitopes of recombinant M. haemolytica leukotoxin as a vaccine or vaccine component against shipping fever.
2. Background
BRD is the major cause of beef cattle morbidity and mortality and of economic losses to the beef cattle industry. The cost of BRD to beef cattle producers approaches $1 billion annually. BRD arises from the interaction of numerous contributing factors including physical stresses associated with weaning, shipment, inclement weather, and overcrowding coupled with viral and bacterial infections. The end result in severe cases is colonization of the lungs with pathogenic bacteria resulting in severe pneumonia. Pasteurella multocida, Haemophilus somnus and Mannheimia (formerly Pasteurella) haemolytica are associated with bovine pneumonia.
However, Mannheimia haemolytica serotype 1(S1) is by far the most important and commonly isolated bacterial pathogen in development of the often-fatal fibrinous pleuropneumonia in beef cattle known as pneumonic pasteurellosis or “shipping fever”.
Prevention and control of shipping fever in feedlots is currently partially addressed by three different mechanisms: antibiotic treatment upon arrival of cattle at the feedlot, antibiotic therapy for sick cattle, and vaccination against BRD viruses and M. haemolytica. The extensive use of antibiotics to control shipping fever increases the possibility of antibiotic residues in meat and the development of drug-resistant bacteria in cattle, including those bacteria with potential impact on human health such as Salmonella and Escherichia coli 0157:H7.
Viral and bacterial vaccines for the control of shipping fever have been used for many years. Despite their availability, the disease continues to be a major bovine health problem. Attempts to develop a vaccine include the following:    U.S. Pat. No. 5,055,400 to Lo et al., (Oct. 8, 1991) discloses the Pasteurella haemolytica leukotoxin gene and protein.    U.S. Pat. No. 5,476,657 (Potter, Dec. 19, 1995) and U.S. Pat. No. 5,871,750 (Potter, Feb. 16, 1991) disclose vaccines comprising Pasteurella haemolytica leukotoxin or truncated forms of the leukotoxin.    U.S. Pat. No. 5,708,155 (Potter et al., Jan. 13, 1998) and U.S. Pat. No. 6,797,272 (Potter et al., Sep. 28, 2004) disclose vaccines comprised of chimeras of Pasteurella haemolytica leukotoxin and an antigen such as somatostatin (SRIF), gonadotropin releasing hormone (GnRH), or rotavirus viral protein 4 (VP4).    U.S. Pat. No. 5,238,823 (Potter et al., Aug. 24, 1993), U.S. Pat. No. 5,273,889 (Potter et al., Dec. 28, 1993), U.S. Pat. No. 5,594,107 (Potter et al., Jan. 14, 1997) and U.S. Pat. No. 6,096,320 (Potter et al., 5 Aug. 1, 2000), disclose vaccines of chimeric proteins comprising Pasteurella haemolytica leukotoxin or an antigenic fragment thereof, and a cytokine such as gamma-interferon or interleukin-2.    U.S. Pat. No. 5,871,750 (Potter, Feb. 16, 1999) discloses vaccine compositions comprising a truncated Pasteurella haemolytica leukotoxin. Other Pasteurella haemolytica cell surface antigenic proteins are also disclosed (fimbrial protein, plasmin receptor protein, and 50K outer membrane protein.    U.S. Pat. No. 5,723,129 (Potter et al., Mar. 3, 1998), U.S. Pat. No. 5,837,268 (Potter et al., Nov. 17, 1998), U.S. Pat. No. 6,022,960 (Potter et al., Feb. 8, 2000) and U.S. Pat. No. 6,521,746 (Potter et al., Feb. 18, 2003) disclose vaccines comprised of chimeras of leukotoxin and gonadotropin releasing hormone (GnRH) multimers.    U.S. Pat. No. 6,475,754 (Bemis et al., Nov. 5, 2002) discloses an antigenic chimeric protein comprising fimbrial protein of Bordetella bronchiseptica and leukotoxin of M. haemolytica. 
Immunity against M. haemolytica is thought to be primarily through production of serum antibodies that neutralize the secreted leukotoxin (LKT) and antibodies against surface antigens. The specific surface antigens that are important in stimulating host immunity to M. haemolytica are not known; however, several studies point towards the importance of outer membrane proteins (OMPs). Pandher et al. demonstrated 21 surface-exposed immunogenic outer membrane proteins in M. haemolytica S1 using protease treatment and Western blotting. (Pandher K, Murphy G L, Confer A W. Identification of immunogenic, surface-exposed outer membrane proteins of Pasteurella haemolytica serotype 1. Vet Microbiol 1999; 65: 215-26) High antibody responses to outer membranes, as measured by ELISA, and to several specific OMPs, as measured by quantitative Western Blotting, consistently correlated with resistance to challenge with virulent M. haemolytica S1 (Confer A W, McCraw R D, Durham J A, Morton R J, Panciera R J. Serum antibody responses of cattle to iron-regulated outer membrane proteins of Pasteurella haemolytica A1. Vet ImmunolImmunopathol 1995; 47: 101-10 and Mosier D A, Simons K R, Confer A W, Panciera R J, Clinkenbeard K D. Pasteurella haemolytica antigens associated with resistance to pneumonic pasteurellosis. Infect Immun 1989; 57: 711-6). Vaccination of cattle with OMP-enriched cellular fractions, from M. haemolytica S1 significantly enhanced resistance of cattle against experimental challenge in the absence of antibodies to LKT. (Morton R J, Panciera R J, Fulton R W, Frank G H, Ewing S A, Homer J T, Confer A W. Vaccination of cattle with outer membrane protein-enriched fractions of Pasteurella haemolytica and resistance against experimental challenge exposure. Am J Vet Res 1995; 56: 875-879) However, the extraction procedure for bacterial outer membranes is time consuming and expensive, making use of purified OMPs as a component of a M. haemolytica vaccine impractical due to cost. Thus, it can be appreciated that the identification of specific, surface exposed immunogenic M. haemolytica OMPs that would stimulate strong antibody responses is highly desirable. Cloning and expression of the appropriate gene(s) and production of recombinant OMP could then be achieved inexpensively.
One of the M. haemolytica OMPs to which high antibody responses correlated with resistance against experimental challenge is a major 45 kDa OMP. Prior studies were undertaken to clone and characterize that protein. In 1998, Pandher et al. reported the cloning, sequencing and characterization of the gene for the major 45-kDa M. haemolytica S1 outer membrane lipoprotein, designated PlpE. (Pandher K, Confer A W, Murphy G L. Genetic and immunologic analyses of PlpE, a lipoprotein important in complement-mediated killing of Pasteurella haemolytica serotype 1. Infect Immun 1998; 66: 5613-9, which publication is incorporated herein by reference) PlpE was found genetically to have 32-35% similarity to an immunogenic lipoprotein, OmIA, demonstrated in Actinobacillus pleuropneumoniae serotypes 1 and 5. Affinity-purified, anti-PlpE antibodies recognized an OMP in all serotypes of M. haemolytica except in serotype 11. In addition, PlpE was determined to be surface-exposed, and in complement-mediated killing assays, a significant reduction was observed in killing of M. haemolytica when bovine immune serum that was depleted of anti-PlpE antibodies was used as the source of antibody, suggesting that antibodies against PlpE may contribute to host defense against the bacterium.
Because of the economic constraints of the cattle industry, bovine vaccines must be low in cost. Therefore, current M. haemolytica vaccines are crude, usually consisting of a culture supernatant, which contains M. haemolytica leukotoxin and sloughed surface proteins, and/or the killed bacterium. Perino and Hunsaker reviewed published field studies on commercial M. haemolytica vaccines and found that efficacy could be established in only 50% of the trials. (Bov Practitioner 1997; 31: 59-66). There is thus an ongoing need for improvement in M. haemolytica vaccines, and for the development of improved methods and compositions for protecting cattle against shipping fever.